An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies

J Immunol Methods. 1988 Aug 9;112(1):63-9. doi: 10.1016/0022-1759(88)90034-8.


An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / isolation & purification*
  • Antibody Specificity*
  • Binding Sites, Antibody
  • Chromatography, Affinity / methods*
  • Cross-Linking Reagents*
  • Female
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Humans
  • Immunoglobulin G / isolation & purification
  • Immunoglobulin G / metabolism
  • Immunohistochemistry / methods
  • Rabbits


  • Antibodies, Monoclonal
  • Cross-Linking Reagents
  • Immunoglobulin G
  • Glyceraldehyde-3-Phosphate Dehydrogenases